This invention relates to liposomes (closed lipid vesicles) made from archaeobacterial lipids, from non-archaeobacterial lipids, and mixtures thereof, and to the use of such liposomes for the enhanced delivery of pharmaceutical and other compounds to specific cell types such as macrophages/phagotocytes/antigen processing cells and to specific tissues in life-forms such as humans, and for the enhancement of the immune response to antigen(s) presented to a life-form such as a human. The vesicles of this invention may be used in vaccine formulations after encapsulation of, or in conjunction with, one or more immunogen, with or without mediation by the presence of other adjuvants or compounds. The invention may also be used, without limitations, for delivery of drugs, antibiotics, pharmaceuticals, biological compounds such as enzymes or DNA or hormones, therapeutics, imaging agents etc to specific cell types or specific tissues in an animal such as a human and other life-forms. Another application may be to use antibiotics/antiviral agents encapsulated in archaeosomes to treat diseases, where the infective organisms may reside as intracellular reservoirs (such as in macrophages) for re-infection.
Liposomes are closed lipid vesicles containing an entrapped aqueous volume. The hydrophilic head groups of the lipids forming liposomes are oriented towards the aqueous environments present inside and outside the liposomes, whereas the hydrophobic regions of the lipids are sandwiched between the polar head groups and away from the aqueous environments. Liposomes may be unilamellar containing a single lipid bilayer, or multilamellar containing multiple bilayers (onion-like in structure) with an aqueous space separating each bilayer from the other. Various techniques for forming liposomes have been described in the literature, including but not limited to, pressure extrusion, detergent dialysis, dehydration-rehydration, reverse-phase evaporation, remote loading, sonication and other methods (13). Liposomes made from conventional ester phospholipids such as phosphatidylcholine are referred to herein as conventional liposomes, even if they contain sterols or other compounds in their bilayer.
Liposomes consisting of a lipid bilayer, a monolayer or a combination thereof, made from any lipid(s) which include in their composition ether lipids extracted from or found in Archaeobacteria, or those synthesized to mimic lipids found in archaeobacteria, are referred to herein as archaeosomes.
Archaea (Archaeobacteria) are considered to be distinct from eubacteria and eukaryotes, and they include aerobic, anaerobic, thermophilic, extremely thermophilic, thermoacidophilic, and halophilic microorganisms. Total lipid extracts from individual species of Archaea consist of polar ether lipids and from 5 to 20% neutral lipids. The polar ether lipids of Archaea consist of branched phytanyl chains which are usually saturated and are attached via ether bonds to the glycerol carbons at the sn-2,3 positions (8). In contrast to this, in conventional phospholipids found in Eubacteria and Eukaryotes, fatty acyl chains which may be unsaturated, are attached via ester bonds to the sn-1,2 carbons of the glycerol backbone. The core structures of the archaeobacterial ether lipids (the polar head groups removed by hydrolysis) consist of the standard diether lipid (2,3-di-O-phytanyl-sn-glycerol or archaeol), and the standard tetraether lipid (2,2xe2x80x2,3,3xe2x80x2-tetra-O-dibiphytanyl-sn-diglycerol or caldarchaeol) and modifications thereof (8). Diether lipids are monopolar like the conventional phospholipids, whereas the tetraether lipids are bipolar. The polar head groups, attached to the sn-1 glycerol carbon in the diethers and to the sn-1 and sn-1xe2x80x2 glycerol carbons in the tetraethers, can vary and may include phospho groups, glyco groups, phosphoglyco groups, polyol groups, c hydroxyl groups (18). In contrast to the phosphatidylcholine conventional lipid commonly used in liposome formulations, the phosphocholine head group is very rarely found in archaeobacterial polar lipids. Archaea provide a large selection of lipids to screen for the preparation of vesicles having the properties useful for specific applications, and overcome the difficulties associated with conventional lipids such as low adjuvanticity and instability.
There is much interest in the use of liposomes for medical, pharmaceutical, and other commercial applications. Most of the research reported on liposomes to-date, has been conducted using conventional phospholipids sometimes mixed with sterols (e.g., cholesterol) or other compounds to improve stability, rather than using either archaeobacterial or non-archaeobacterial ether lipids.
In a comparative study on the uptake of liposomes made with 1,2-diacyl-sn-glycero-3-phosphocholine and its ether analog, by cultured rat liver hepatocytes, the cellular uptake of both liposome types was found to be similar (21). In another study, liposomes made with either dipalmitoyl phosphatidylcholine or its ether analogue 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine, were phagocytosed at about the same rate by J774.E1 macrophage cells (6). Therefore, from these disclosures it would be expected that liposomes made with ether lipids, including by extension those from ether lipids either extracted from Arcaea or from ether lipids chemically synthesized to mimic the unique lipid structures of Archaea, would be taken up by certain cells such as macrophages, to a similar extent as conventional liposomes. However, the current invention proves to the contrary, showing enhanced phagocytosis of vesicles (archaeosomes) made with archaeobacterial ether lipids.
Intracellular delivery of antibiotics and other drugs to control pathogens which reside within certain cell types such as macrophages is a current problem, e.g., the bacterium Mycobacterium tuberculosis which causes tuberculosis, viruses such as the human immunodeficiency virus (HIV) which causes acquired immune deficiency syndrome (AIDS), and parasites which cause malaria. A superior uptake of archaeosomes made with ether lipids of Archaea would have commercial utility in enhancing the delivery of drugs, antigens, and other compounds targeted for delivery to phagocytic cells of a life-form, such as a human.
There is considerable interest in the potential use of liposomes in the field of vaccine applications. Liposomes prepared from conventional phospholipids, sometimes mixed with cholesterol or other compounds (conventional liposomes) have been tested as potential antigen carriers/vehicles. Allison and Gregoriadis (1) reported that liposomes prepared from egg phosphatidylcholine had some adjuvant activity, provided a negatively charged lipid was included in the liposome composition. Since conventional liposomes often demonstrate only small adjuvant effects as compared with administration of the free antigen, various immunostimulatory substances such as lipid A have been co-incorporated into the liposomes, together with the antigen (4). However, as is the case with lipid A and Freund""s adjuvant, immunostimulatory substances may have toxicity associated problems, making them unsuitable for vaccine applications.
The humoral immune response, in mice, to bovine serum albumin encapsulated in liposomes made with dialkyl-ether sn-3-phosphatidylcholine was lower than that obtained with similar liposomes made with diacyl-ester sn-3 phosphatidylcholine (17). There is no teaching in the prior art to suggest true compared with liposomes made using conventional phospholipids, those made using archaeobacterial ether lipids would have a superior adjuvant effect in stimulating the immune response to an antigen administered into an animal by various routes (including but not limited to intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.), and peroral(p.o.)).
Coenzyme Q10 (also known as CoQ10, ubiquinone-10 or ubidecarenone-10) is present in mammalian cells having mitochondria where it is a redox component in the respiratory chain. Since decreased levels of CoQ10 have been implicated in certain pathological conditions such as myocardiac insufficiency, cardiac infarction, muscular dystrophy, arterious hypertension, and in the symptoms of ageing, it has been considered for therapeutic applications in such cases. Deficiency of CoQ10 has also been reported in AIDS patients (5). CoQ10 is extremely hydrophobic in nature and unless its chemical structure is artificially modified, it is not soluble in aqueous buffers or water. Similarities are noted between CoQ10 and vitamin E, with respect to serving as immune stimulators and as anti-oxidants. CoQ10 as an emulsion with detergents has been shown to enhance the in vivo phagocytic activity in animal models (3). Labelled CoQ10 has been used in conventional liposomes as a marker for myocardial imaging and for studying tissue distribution of conventional liposomes coated with polysaccharides (7,22). In liposomes, CoQ10 is associated with the lipid layer of the vesicles. However, none of these or other prior art publications teach that the combination of CoQ10 in archaeosomes would enhance the phagocytosis of the resultant vesicles by macrophage cells (compared to that of the archaeosomes without CoQ10), or that such a combination would allow the alteration in targeting profiles to specific tissues when the vesicles are administered to an animal via different routes, or that such combinations would further enhance the immune response to co-administered immunogen(s) (current claimed invention). Similarly, the prior art does not teach that the combination of CoQ10 in conventional liposomes would increase the phagocytosis of the resultant liposomes by macrophages, or allow for the alteration of tissue targeting profiles when the liposomes are administered to an animal by different routes, or that liposomal CoQ10 combination would enhance the immune response to co-administered immunogen(s) compared to the liposomal immunogen in the absence of CoQ10.
It is an object of this invention to use archaeosomes as beneficial carriers of antigens, immunogenic compounds, DNA, drugs, therapeutic compounds, pharmaceutical compounds, imaging agents or tracers, and to deliver these to specific cells such as the macrophages or to specific tissues, in life-forms such as the human.
It is a further object of this invention to provide archaeosomes that have enhanced adjuvant activity for the generation of an immune response to an immunogen, and for vaccine applications in an animal such as a human, wherein the antigen(s) can be encapsulated in, can be associated with, or not associated with the archaeosomes, at the time of administration via different routes. In some instances the immune response (the level of response and its duration) to an immunogen delivered via an archaeosome being comparable to that obtained with Freund""s adjuvant as the immunostimulator.
Yet another object of the invention is to use archaeosomes prepared with lipids containing a high proportion of tetraether bipolar lipid(s) that are found in, or mimic those found in, members of archaeobacteria, to enhance and prolong the immune response to an immunogen that is either codministered as part of the archaeosome or administered at the same time as the archaeosome, into an animal.
It is another object of the invention to incorporate coenzyme Q10 into archaeosomes, or into liposomes prepared exclusively from lipids other than archaeobacterial-like ether lipids, to enhance the phagocytosis of the respective archaeosomes/liposomes, and/or to enhance the delivery of CoQ10 as well as other associated drug(s), and to enhance the immune response to an antigen associated with the respective archaeosomes/liposomes.
It is another object of this invention to incorporate CoQ10 into archaeosomes and conventional liposomes, sometimes in combination with polyethylene glycol lipid conjugates, to increase the delivery of various associated compounds to specific organ tissues when the respective vesicles are administered to an animal via various routes such as p.o., i.m., i.v., s.c, and i.p. The combination of CoQ10 in archaeosomal or conventional liposomal vesicles, including vesicles that may have been sterically stabilized by association with polyethyleneglycol conjugates, would therefore further increase the utility of archaeosomes and of conventional liposomes, for delivery of compounds, including immunogens and CoQ10 itself, to phagocytic cells and to specific tissues.
It is yet another object of the invention to incorporate optimal amounts of archaeobacterial ether lipid(s) into mixtures with conventional lipids to prepare vesicles that have the above improved characteristics.
All of the above aspects of the current invention are interrelated in concept.